1. Field of the Invention
This invention relates to buffering compositions, and more particularly, to buffering compositions useful for immunological and immunochemical assay components, such as antibodies and antigens.
2. Discussion of the art
Many types of immunoassays can be run on an apparatus of the type described in U.S. Pat. No. 5,358,691, incorporated herein by reference. In general, immunoassays can be classified into two major categories--homogeneous and heterogeneous. Homogeneous and heterogeneous immunoassays depend upon the ability of a first binding member of a binding member pair, e.g., an antigen, to specifically bind to a second binding member of a binding member pair, e.g., an antibody. A conjugate, comprising one of such binding members labeled with a detectable moiety, is employed to determine the extent of such binding. For example, such binding member pairs can be an antigen and an antibody to such antigen. The conjugate, which can comprise the antigen, either participates in a binding reaction with the antibody or does not participate in such a reaction. The amount of detectable moiety detected and measured after the reaction can be correlated to the amount of antibody present in the test sample.
Heterogeneous assays can be performed in a competitive immunoassay format or in a sandwich immunoassay format. In the competitive immunoassay format, an antigen can be immobilized to a solid phase material whereby the amount of detectable moiety that is bound to a solid phase material can be detected, measured, and correlated to the amount of antibody present in the test sample. Examples of solid phase materials include beads, particles, microparticles, and the like. In the sandwich immunoassay format, a test sample containing, for example, an antibody is contacted with a protein such as an antigen. The antigen is immobilized on a solid phase material. Examples of solid phase materials include beads, particles, microparticles, and the like. The solid phase material is typically treated with a second antigen or antibody that has been labeled with a detectable moiety. The second antigen or antibody then becomes bound to the corresponding antigen or antibody on the solid phase material and, after one or more washing steps to remove any unbound material, an indicator material, such as a chromogenic substance, is introduced to react with the detectable moiety to produce a detectable signal, e.g., a color change. The color change is then detected, measured, and correlated to the amount of antibody present in the test sample. It should also be noted that various diluents and buffers are also required to optimize the operation of the microparticles, antigens, conjugates, and other components of the assay that participate in chemical reactions.
A heterogeneous immunoassay that can be performed with the apparatus of U.S. Pat. No. 5,358,691 in either a competitive or sandwich immunoassay format is a microparticle capture enzyme immunoassay, such as that described in Clinical Chemistry, Volume 34, No. 9, pages 1726-1732 (1988), employing microparticles as the solid phase material. This article is incorporated herein by reference.
A step-by-step description of a microparticle capture enzyme immunoassay procedure is set forth at col. 35, line 60 through col. 44, line 22 of U.S. Pat. No. 5,358,691.
Various components of of kits used in immunoassays run on the apparatus described in U.S. Pat. No. 5,358,691 must fulfill certain functional requirements. The microparticles must provide a solid phase for antibody capture, serving as a support for the antigens used for said capture. These particles must themselves be captured, e.g., by a matrix cell filter, to permit detection of bound antibody by the conjugate/substrate combination. The microparticle diluent must provide a medium that maintains the antigens' ability to be recognized by complementary antibodies in patient specimens (stability) while not inhibiting that recognition (signal generation). The conjugate must provide a means to specifically recognize antibodies bound to antigens on the microparticle and a means to generate a signal to indicate the presence of conjugate on the microparticle. The conjugate diluent must provide a medium that preserves and optimizes the ability of one binding pair member on the conjugate to recognize its complementary binding pair member, while simultaneously preserving and optimizing the ability of the enzyme portion, e.g., alkaline phosphatase portion, to hydrolyze the substrate into a detectable entity. In addition, the conjugate diluent must contain components that will minimize, if not eliminate, nonspecific binding of the conjugate to the matrix or microparticle, thus preventing the generation of a false signal. The sample dilution buffer must provide a medium that optimizes the ability of antibodies in a specimen to bind to antigen on the microparticles while preventing nonspecific interactions that might lead to the generation of a false signal
Because certain antigens used in immunoassays are unstable after prolonged exposure to heat, it would be desirable to increase the stability of antigens to heat stress. Because antigens are characterized by certain epitopes, it would be desirable to increase the stability of the antigens by increasing the stability of the characteristic epitopes.